The goal of this proposal is further characterize the similarities and differences of two prenlytransferases, farnesyltransferase (FTase) and geranylgeranyltransferase type I (GGTaseI). In vivo, these enzymes are responsible for prenylating many protein substrates including Ras proteins. The discovery that prenylation was critical for the transforming abilities of oncogenic Ras proteins makes prenyltransferases a key target for anticancer therapeutics. In-depth information on the catalytic mechanisms of these proteins will aid in the development and refinement of substrate- based inhibitors. The transition-state for the reaction catalyzed by FTase has characteristics of both a nucleophilic and electrophilic mechanism. To distinguish between these mechanisms, transient heavy atom isotope effects will be studied. If a carbocation intermediate is relevant in the FTase mechanism, both primary and secondary isotope effects should be detected. This proposal will also investigate the role of pyrophosphate (PPi) release on the overall, steady-state kcat of the base reaction. This approach will involve the combination of site-directed mutagenesis and the implementation of an assay designed to measure PPi production directly. Finally, this proposal will investigate the metal coordination and catalytic mechanism of GGTase I for comparison to those features of the related FTase.